CN113444745A - Construction of virus S protein mutant pseudovirus disc and vaccine evaluation application thereof - Google Patents
Construction of virus S protein mutant pseudovirus disc and vaccine evaluation application thereof Download PDFInfo
- Publication number
- CN113444745A CN113444745A CN202110573556.XA CN202110573556A CN113444745A CN 113444745 A CN113444745 A CN 113444745A CN 202110573556 A CN202110573556 A CN 202110573556A CN 113444745 A CN113444745 A CN 113444745A
- Authority
- CN
- China
- Prior art keywords
- pseudovirus
- protein
- cells
- vaccine
- disc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241001112090 Pseudovirus Species 0.000 title claims abstract description 103
- 101710139375 Corneodesmosin Proteins 0.000 title claims abstract description 61
- 102100031673 Corneodesmosin Human genes 0.000 title claims abstract description 59
- 229960005486 vaccine Drugs 0.000 title claims abstract description 33
- 241000700605 Viruses Species 0.000 title claims abstract description 19
- 238000010276 construction Methods 0.000 title claims abstract description 16
- 238000011156 evaluation Methods 0.000 title claims abstract description 16
- 230000035772 mutation Effects 0.000 claims abstract description 50
- 230000000694 effects Effects 0.000 claims abstract description 20
- 208000015181 infectious disease Diseases 0.000 claims abstract description 13
- 229940022962 COVID-19 vaccine Drugs 0.000 claims abstract description 11
- 241000711573 Coronaviridae Species 0.000 claims abstract description 11
- 230000001900 immune effect Effects 0.000 claims abstract description 5
- 239000013613 expression plasmid Substances 0.000 claims description 25
- 108060001084 Luciferase Proteins 0.000 claims description 18
- 210000002966 serum Anatomy 0.000 claims description 16
- 241000713772 Human immunodeficiency virus 1 Species 0.000 claims description 15
- 239000013612 plasmid Substances 0.000 claims description 15
- 239000001963 growth medium Substances 0.000 claims description 14
- 241001678559 COVID-19 virus Species 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
- 101710091045 Envelope protein Proteins 0.000 claims description 9
- 101710188315 Protein X Proteins 0.000 claims description 9
- 238000010790 dilution Methods 0.000 claims description 9
- 239000012895 dilution Substances 0.000 claims description 9
- 238000002255 vaccination Methods 0.000 claims description 9
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 0.000 claims description 7
- 150000001413 amino acids Chemical class 0.000 claims description 7
- 238000006386 neutralization reaction Methods 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 7
- 102000053723 Angiotensin-converting enzyme 2 Human genes 0.000 claims description 6
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 claims description 6
- 239000005089 Luciferase Substances 0.000 claims description 6
- 230000000295 complement effect Effects 0.000 claims description 6
- 239000003623 enhancer Substances 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 6
- 230000003993 interaction Effects 0.000 claims description 6
- 238000002372 labelling Methods 0.000 claims description 6
- 239000013641 positive control Substances 0.000 claims description 6
- 229920000209 Hexadimethrine bromide Polymers 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 5
- 230000008859 change Effects 0.000 claims description 5
- 239000013642 negative control Substances 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- HBZBAMXERPYTFS-SECBINFHSA-N (4S)-2-(6,7-dihydro-5H-pyrrolo[3,2-f][1,3]benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid Chemical compound OC(=O)[C@H]1CSC(=N1)c1nc2cc3CCNc3cc2s1 HBZBAMXERPYTFS-SECBINFHSA-N 0.000 claims description 3
- 108020004705 Codon Proteins 0.000 claims description 3
- 108700008625 Reporter Genes Proteins 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 239000007795 chemical reaction product Substances 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 238000003259 recombinant expression Methods 0.000 claims description 3
- 230000006798 recombination Effects 0.000 claims description 3
- 238000005215 recombination Methods 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 230000009385 viral infection Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims 1
- 230000008569 process Effects 0.000 description 5
- 230000009471 action Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 3
- 230000004544 DNA amplification Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000029578 entry into host Effects 0.000 description 2
- 230000005745 host immune response Effects 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 102000029301 Protein S Human genes 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 101710198474 Spike protein Proteins 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000036438 mutation frequency Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16041—Use of virus, viral particle or viral elements as a vector
- C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Virology (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses construction of a virus S protein mutant pseudovirus disc and application of the pseudovirus disc in vaccine evaluation, which comprises the following steps: step one, constructing a pseudovirus system; step two, detecting the activity of the pseudovirus; step three, the pseudovirus is used for evaluating the immune effect of the new corona vaccine; in the first step, according to the genome sequence number GenBank of the novel coronavirus, MN908947, mutation sites with more strain numbers and wider propagation range of the mutation sites in the database are analyzed and counted, and 40 mutation sites are selected for constructing the pseudovirus; detecting the activity of the S protein pseudovirus in the step two, and forming a new coronavirus pseudovirus disk after classification and statistics; the invention can more comprehensively evaluate the immune effect of the new corona vaccine, particularly evaluate whether the current new corona vaccine can effectively prevent the infection and the prevalence of the new corona virus mutant strain when the false virus disk is applied to the effect evaluation of the SARS-CoV-2 vaccine in the third step.
Description
Technical Field
The invention relates to the technical field of vaccine evaluation, in particular to construction of a virus S protein mutant pseudovirus disc and application of the virus S protein mutant pseudovirus disc to vaccine evaluation.
Background
The invasion of host cells by the novel coronavirus SARS-CoV-2 is mainly mediated by the spike protein S, and the mutation of the S protein not only affects the replication ability and infection ability of the virus, but also can cause the change of host immune response. Although the mutation frequency of SARS-CoV-2 is lower than that of other RNA viruses without reverse transcription correction function, there is currently no clear conclusion on how these mutation sites affect virus invasion into host cells. In addition, Antigenic drift (Antigenic drift) may occur due to virus surface glycoprotein mutation, and the cross-immune protection effect of SARS-CoV-2 vaccine derived from parent strain is unknown. The traditional vaccine effect detection means is determined by using competitive ELISA or single pseudovirus neutralization test of hACE2 and RBD at present; meanwhile, the S protein is used as a membrane protein of coronavirus and is firstly influenced by the immune pressure of a host in the process of transmitting the virus to a human. Conversely, mutations in the S protein will also tend to cause a change in the host immune response. Currently, the global spread of SARS-CoV-2 results in the production of a large number of mutant strains. Although the research results of SARS-CoV-2 vaccine are consortious recently, the continuously generated mutant strain puts higher requirements on vaccine design, a comprehensive vaccine evaluation system is lacked under the background that the epidemic situation is global and the strain is rapidly mutated, and the detection standard of effectiveness after vaccination is not clearly defined, so that the construction of the virus S protein mutant pseudovirus disk and the application of the vaccine evaluation are necessary.
Disclosure of Invention
The invention aims to provide the construction of a virus S protein mutant pseudovirus disc and the evaluation application of a vaccine thereof, so as to solve the problems in the background technology.
In order to solve the technical problems, the invention provides the following technical scheme: the construction of the virus S protein mutant pseudovirus disc and the vaccine evaluation application thereof comprise the following steps: step one, constructing a pseudovirus system; step two, detecting the activity of the pseudovirus; step three, the pseudovirus is used for evaluating the immune effect of the new corona vaccine;
in the first step, according to the analysis data of the mutation sites of the SARS-CoV-2S protein gene published by the national biological information center 2019 novel coronavirus information library, the mutation of the S protein is mainly divided into ACE2 binding region-non-key interaction amino acid, ACE2 binding region-key interaction amino acid and non-ACE 2 binding region, then the mutation sites with more strain number and wider spread range in the database are selected for statistics, and according to the novel coronavirus genome sequence number GenBank: MN908947, the codon optimizes and synthesizes the full-length gene sequence of the S protein and connects with the eukaryotic expression plasmid pcDNA3.1(+) to obtain the recombinant expression plasmid pcDNA3.1(+) -Spike; when constructing S protein mutant, adopting point mutation mode to make reverse PCR on eukaryotic expression plasmid pcDNA3.1(+) -Spike, the 5' end of forward and reverse amplification primer contains 15-21bp reverse complementary region, when designing primer, the introduced mutation is contained in complementary region; digesting the product after amplifying the target plasmid, and performing recombination reaction after removing the methylated template plasmid; converting the reaction product, cloning and identifying, wherein the S protein mutant expression plasmid with successful mutation is obtained after the sequencing is correct; the S protein mutant expression plasmid and HIV-1 skeleton plasmid pNL4-3 Luc + R-E- (luciferase gene replaces Env envelope protein gene of HIV-1) are respectively co-transfected into 293T cells to obtain S protein mutant pseudoviruses; meanwhile, co-transfecting 293T cells with an S protein expression plasmid without mutation and pNL4-3 Luc + R-E- (luciferase gene replacing Env envelope protein gene of HIV-1) to obtain a novel coronavirus S protein pseudovirus;
in the second step, the constructed expression plasmids of different S protein mutation sites are respectively co-transfected with an HIV-1 framework plasmid pNL4-3 Luc + R-E- (luciferase gene replaces Env envelope protein gene of HIV-1) to 293T cells to package S protein pseudoviruses of different mutation sites, and the mutated pseudoviruses are collectively called a SARS-CoV-2S protein pseudovirus disk; because the pNL4-3 Luc + R-E-plasmid is provided with a reporter gene for expressing luciferase, the pseudovirus disc can express the luciferase gene in cells after infecting susceptible cells; therefore, after the luciferase substrate is added, RLU in cells is detected according to a chemiluminescence apparatus, and the infection capacity of the pseudoviruses at different mutation sites can be judged; before the construction of a pseudovirus disc, a SARS-CoV-2 susceptible cell line HEK293T/hACE2 has been successfully constructed; after the S protein pseudovirus disc was successfully constructed, we examined the ability of the pseudovirus disc to infect HEK293T/hACE2 cells: HEK293T/hACE2 cells at 1X 10 in 24h in advance4The density of each hole is paved on a 96-hole plate, the titrated different S protein pseudoviruses are standardized on the 2 nd day and then added into the 96-hole plate, and a complete culture medium without the pseudoviruses is set as a negative control; 50 μ l of pseudovirus group was used as positive control, while 5 μ g/mL of polybrene as infection enhancer was added; after the pseudovirus infects the cells for 6h, the complete culture medium is replaced, and after the pseudovirus infects the cells for 48h, the RLU is detected by using a chemiluminescence enzyme-labeling instrument; classifying different mutants in a pseudovirus disc according to the magnitude order of RLU, namely the infection capacity of susceptible cells, analyzing the reasons of the change of the virus infection capacity caused by the site mutation and finding out the main site acting with a receptor;
in the third step, the SARS-CoV-2 vaccine is currently or gradually subjected to clinical tests at various stages in China, serum of people who are inoculated with various brands of vaccines is collected, the time of blood sample collection and distance vaccination is recorded as 0-12 months, and serum of people who are not inoculated is collected for clinical testsNegative control; taking 50 mul of pseudovirus group as positive control, testing the neutralization experiment result of collected serum by a pseudovirus tray, and comprehensively evaluating the cross immune protection effect of each vaccine on SARS-CoV-2 after mutation; in addition, the time for maintaining the antibody generated in the body of the vaccinated population is determined according to the collection time of each sample, and theoretical reference is provided for the subsequent vaccination scheme of the SARS-CoV-2 vaccine; the neutralization experimental procedure was as follows: HEK293T/hACE2 cells at 1X 10 in 24h in advance4The serum after the second vaccination of the vaccine is diluted by a complete culture medium according to a 2-fold gradient in the following day, the initial dilution is 1:2, 8 gradients (1: 2 to 1:256) are set, the diluted serum is mixed with 50 microliter of titrated pseudovirus in equal volume, the mixed serum is added into a 96-well plate which is paved with HEK293T/hACE2 cells in advance after 1h incubation at 37 ℃, 50 microliter of complete culture medium without pseudovirus and 50 microliter of pseudovirus are set as a control, and 5 microliter/mL of infection enhancer polybrene is added at the same time; after the pseudovirus infects the cells for 6h, the complete culture medium is replaced, and after the pseudovirus infects the cells for 48h, the RLU is detected by using a chemiluminescence enzyme-labeling instrument; the cross immune protection effect of the vaccine on different mutant strains can be effectively evaluated according to the detected RLU value.
According to the above technical solution, in the second step, the RLU is called Relative luciferase activity.
According to the technical scheme, in the third step, the initial dilution multiple is 1:2, the dilution range is 1:2 to 1:256, and 8 gradients are arranged in 3 parallels.
Compared with the prior art, the invention has the following beneficial effects: the method for evaluating the effect of the new corona vaccine by using the S protein pseudovirus directly performs mutation construction in eukaryotic expression plasmids, does not need to perform mutation amplification on S protein genes and then connect the expression plasmids, simultaneously avoids mismatching in the S protein gene amplification process, constructs various mutant new corona pseudoviruses after performing classification statistics on main mutation sites of the S protein, and applies the formed pseudovirus disk to the effect evaluation of SARS-CoV-2 vaccine; therefore, the invention is beneficial to more fully cover the current new crown mutant strain.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a flow chart of a method of the present invention;
FIG. 2 is a statistical diagram of the amino acid mutation sites of SARS-CoV-2S protein in the present invention;
FIG. 3 is a technical roadmap for the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-3, the present invention provides a technical solution: the construction of the virus S protein mutant pseudovirus disc and the vaccine evaluation application thereof comprise the following steps: step one, constructing a pseudovirus system; step two, detecting the activity of the pseudovirus; step three, the pseudovirus is used for evaluating the immune effect of the new corona vaccine;
in the first step, according to the analysis data of the mutation sites of the SARS-CoV-2S protein gene published by the national biological information center 2019 novel coronavirus information library, the mutation of the S protein is mainly divided into ACE2 binding region-non-key interaction amino acid, ACE2 binding region-key interaction amino acid and non-ACE 2 binding region, then the mutation sites with more strain number and wider spread range in the database are selected for statistics, and according to the novel coronavirus genome sequence number GenBank: MN908947, the codon optimizes and synthesizes the full-length gene sequence of the S protein and connects with the eukaryotic expression plasmid pcDNA3.1(+) to obtain the recombinant expression plasmid pcDNA3.1(+) -Spike; when constructing S protein mutant, adopting point mutation mode to make reverse PCR on eukaryotic expression plasmid pcDNA3.1(+) -Spike, the 5' end of forward and reverse amplification primer contains 15-21bp reverse complementary region, when designing primer, the introduced mutation is contained in complementary region; digesting the product after amplifying the target plasmid, and performing recombination reaction after removing the methylated template plasmid; converting the reaction product, cloning and identifying, wherein the S protein mutant expression plasmid with successful mutation is obtained after the sequencing is correct; the S protein mutant expression plasmid and HIV-1 skeleton plasmid pNL4-3 Luc + R-E- (luciferase gene replaces Env envelope protein gene of HIV-1) are respectively co-transfected into 293T cells to obtain S protein mutant pseudoviruses; meanwhile, co-transfecting 293T cells with an S protein expression plasmid without mutation and pNL4-3 Luc + R-E- (luciferase gene replacing Env envelope protein gene of HIV-1) to obtain a novel coronavirus S protein pseudovirus;
in the second step, the constructed expression plasmids of different S protein mutation sites are respectively co-transfected with an HIV-1 framework plasmid pNL4-3 Luc + R-E- (luciferase gene replaces Env envelope protein gene of HIV-1) to 293T cells to package S protein pseudoviruses of different mutation sites, and the mutated pseudoviruses are collectively called a SARS-CoV-2S protein pseudovirus disk; because the pNL4-3 Luc + R-E-plasmid is provided with a reporter gene for expressing luciferase, the pseudovirus disc can express the luciferase gene in cells after infecting susceptible cells; therefore, the RLU in the cells is detected according to a chemiluminescence apparatus after the luciferase substrate is added, and the RLU is called Relative luciferase activity, so that the infection capacity of the pseudoviruses at different mutation sites can be judged; before the construction of a pseudovirus disc, a SARS-CoV-2 susceptible cell line HEK293T/hACE2 has been successfully constructed; after the S protein pseudovirus disc was successfully constructed, we examined the ability of the pseudovirus disc to infect HEK293T/hACE2 cells: HEK293T/hACE2 cells at 1X 10 in 24h in advance4The density of each hole is paved on a 96-hole plate, the titrated different S protein pseudoviruses are standardized on the 2 nd day and then added into the 96-hole plate, and a complete culture medium without the pseudoviruses is set as a negative control; 50 μ l of pseudovirus group was used as positive control, while 5 μ g/mL of polybrene as infection enhancer was added; after the pseudovirus infects the cells for 6h, the complete culture medium is replaced, and after the pseudovirus infects the cells for 48h, the RLU is detected by using a chemiluminescence enzyme-labeling instrument; the different mutants in the pseudoviral disks were ranked according to the magnitude of RLU, i.e.the infectivity of susceptible cells, and the mutations at these sites were directedAnalyzing the causes of the change of the virus infection capacity and finding out the main sites of the action with the receptor;
in the third step, the SARS-CoV-2 vaccine is currently or gradually subjected to clinical tests at various stages in China, serum of people inoculated with various brands of vaccines is collected, the time of blood sample collection and distance vaccination is recorded as 0-12 months, and serum of people who are not inoculated is collected as negative control; taking 50 mul of pseudovirus group as positive control, testing the neutralization experiment result of collected serum by a pseudovirus tray, and comprehensively evaluating the cross immune protection effect of each vaccine on SARS-CoV-2 after mutation; in addition, the time for maintaining the antibody generated in the body of the vaccinated population is determined according to the collection time of each sample, and theoretical reference is provided for the subsequent vaccination scheme of the SARS-CoV-2 vaccine; the neutralization experimental procedure was as follows: HEK293T/hACE2 cells at 1X 10 in 24h in advance4The serum is diluted by using complete culture medium according to 2-fold gradient with the initial dilution of 1:2, 8 gradients (1: 2 to 1:256) are set in total, the initial dilution is 1:2, the dilution range is 1:2 to 1:256, 8 gradients are set in total, and 3 parallels are set; mixing the diluted serum with 50 mu l of titrated pseudovirus in equal volume, incubating at 37 ℃ for 1h, adding the mixture into a 96-well plate which is paved with HEK293T/hACE2 cells in advance, setting 50 mu l of complete culture medium without pseudovirus and 50 mu l of pseudovirus group as a control, and adding 5 mu g/mL of polybrene as an infection enhancer; after the pseudovirus infects the cells for 6h, the complete culture medium is replaced, and after the pseudovirus infects the cells for 48h, the RLU is detected by using a chemiluminescence enzyme-labeling instrument; the cross immune protection effect of the vaccine on different mutant strains can be effectively evaluated according to the detected RLU value.
Based on the above, the invention has the advantages that the invention constructs new mutant coronavirus after classification statistics is carried out on the main mutation sites of the S protein, and the formed pseudovirus disk is applied to the effect evaluation of SARS-CoV-2 vaccine; the method is favorable for more comprehensively covering the current new crown mutant strain, simultaneously directly carrying out mutation construction in eukaryotic expression plasmids, and connecting the expression plasmids after carrying out mutation amplification on S protein genes, thereby reducing the occurrence of mismatching rate in the S protein gene amplification process.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. The construction of the virus S protein mutant pseudovirus disc and the vaccine evaluation application thereof comprise the following steps: step one, constructing a pseudovirus system; step two, detecting the activity of the pseudovirus; step three, the pseudovirus is used for evaluating the immune effect of the new corona vaccine; the method is characterized in that:
in the first step, according to the analysis data of the mutation sites of the SARS-CoV-2S protein gene published by the national biological information center 2019 novel coronavirus information library, the mutation of the S protein is mainly divided into ACE2 binding region-non-key interaction amino acid, ACE2 binding region-key interaction amino acid and non-ACE 2 binding region, then the mutation sites with more strain number and wider spread range in the database are selected for statistics, and according to the novel coronavirus genome sequence number GenBank: MN908947, the codon optimizes and synthesizes the full-length gene sequence of the S protein and connects with the eukaryotic expression plasmid pcDNA3.1(+) to obtain the recombinant expression plasmid pcDNA3.1(+) -Spike; when constructing S protein mutant, adopting point mutation mode to make reverse PCR on eukaryotic expression plasmid pcDNA3.1(+) -Spike, the 5' end of forward and reverse amplification primer contains 15-21bp reverse complementary region, when designing primer, the introduced mutation is contained in complementary region; digesting the product after amplifying the target plasmid, and performing recombination reaction after removing the methylated template plasmid; converting the reaction product, cloning and identifying, wherein the S protein mutant expression plasmid with successful mutation is obtained after the sequencing is correct; the S protein mutant expression plasmid and HIV-1 skeleton plasmid pNL4-3 Luc + R-E- (luciferase gene replaces Env envelope protein gene of HIV-1) are respectively co-transfected into 293T cells to obtain S protein mutant pseudoviruses; meanwhile, co-transfecting 293T cells with an S protein expression plasmid without mutation and pNL4-3 Luc + R-E- (luciferase gene replacing Env envelope protein gene of HIV-1) to obtain a novel coronavirus S protein pseudovirus;
in the second step, the constructed expression plasmids of different S protein mutation sites are respectively co-transfected with an HIV-1 framework plasmid pNL4-3 Luc + R-E- (luciferase gene replaces Env envelope protein gene of HIV-1) to 293T cells to package S protein pseudoviruses of different mutation sites, and the mutated pseudoviruses are collectively called a SARS-CoV-2S protein pseudovirus disk; because the pNL4-3 Luc + R-E-plasmid is provided with a reporter gene for expressing luciferase, the pseudovirus disc can express the luciferase gene in cells after infecting susceptible cells; therefore, after the luciferase substrate is added, RLU in cells is detected according to a chemiluminescence apparatus, and the infection capacity of the pseudoviruses at different mutation sites can be judged; before the construction of a pseudovirus disc, a SARS-CoV-2 susceptible cell line HEK293T/hACE2 has been successfully constructed; after the S protein pseudovirus disc was successfully constructed, we examined the ability of the pseudovirus disc to infect HEK293T/hACE2 cells: HEK293T/hACE2 cells at 1X 10 in 24h in advance4The density of each hole is paved on a 96-hole plate, the titrated different S protein pseudoviruses are standardized on the 2 nd day and then added into the 96-hole plate, and a complete culture medium without the pseudoviruses is set as a negative control; 50 μ l of pseudovirus group was used as a positive control with the addition ofPolybrene as an infection enhancer at 5. mu.g/mL; after the pseudovirus infects the cells for 6h, the complete culture medium is replaced, and after the pseudovirus infects the cells for 48h, the RLU is detected by using a chemiluminescence enzyme-labeling instrument; classifying different mutants in a pseudovirus disc according to the magnitude order of RLU, namely the infection capacity of susceptible cells, analyzing the reasons of the change of the virus infection capacity caused by the site mutation and finding out the main site acting with a receptor;
in the third step, the SARS-CoV-2 vaccine is currently or gradually subjected to clinical tests at various stages in China, serum of people inoculated with various brands of vaccines is collected, the time of blood sample collection and distance vaccination is recorded as 0-12 months, and serum of people who are not inoculated is collected as negative control; taking 50 mul of pseudovirus group as positive control, testing the neutralization experiment result of collected serum by a pseudovirus tray, and comprehensively evaluating the cross immune protection effect of each vaccine on SARS-CoV-2 after mutation; in addition, the time for maintaining the antibody generated in the body of the vaccinated population is determined according to the collection time of each sample, and theoretical reference is provided for the subsequent vaccination scheme of the SARS-CoV-2 vaccine; the neutralization experimental procedure was as follows: HEK293T/hACE2 cells at 1X 10 in 24h in advance4Density of/well plated in 96-well plates, serum 2 weeks after the second vaccination with vaccine was diluted in complete medium on the next day in a 2-fold gradient with 1:2 initial dilution for a total of 8 gradients (1: 2 to 1: 256); mixing the diluted serum with 50 mu l of titrated pseudovirus in equal volume, incubating at 37 ℃ for 1h, adding the mixture into a 96-well plate which is paved with HEK293T/hACE2 cells in advance, setting 50 mu l of complete culture medium without pseudovirus and 50 mu l of pseudovirus group as a control, and adding 5 mu g/mL of polybrene as an infection enhancer; after the pseudovirus infects the cells for 6h, the complete culture medium is replaced, and after the pseudovirus infects the cells for 48h, the RLU is detected by using a chemiluminescence enzyme-labeling instrument; the cross immune protection effect of the vaccine on different mutant strains can be effectively evaluated according to the detected RLU value.
2. The construction of the virus S protein mutant pseudovirus disc and the vaccine evaluation application thereof according to claim 1, wherein the virus S protein mutant pseudovirus disc comprises the following components: in the second step, the RLU is called Relative luciferase activity.
3. The construction of the virus S protein mutant pseudovirus disc and the vaccine evaluation application thereof according to claim 1, wherein the virus S protein mutant pseudovirus disc comprises the following components: in the third step, the initial dilution multiple is 1:2, the dilution range is 1:2 to 1:256, and 8 gradients are arranged in 3 parallels.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110573556.XA CN113444745A (en) | 2021-05-25 | 2021-05-25 | Construction of virus S protein mutant pseudovirus disc and vaccine evaluation application thereof |
| CN202311245855.6A CN117265014A (en) | 2021-05-25 | 2021-05-25 | Construction method and application of virus S protein mutant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110573556.XA CN113444745A (en) | 2021-05-25 | 2021-05-25 | Construction of virus S protein mutant pseudovirus disc and vaccine evaluation application thereof |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311245855.6A Division CN117265014A (en) | 2021-05-25 | 2021-05-25 | Construction method and application of virus S protein mutant |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113444745A true CN113444745A (en) | 2021-09-28 |
Family
ID=77810162
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311245855.6A Pending CN117265014A (en) | 2021-05-25 | 2021-05-25 | Construction method and application of virus S protein mutant |
| CN202110573556.XA Pending CN113444745A (en) | 2021-05-25 | 2021-05-25 | Construction of virus S protein mutant pseudovirus disc and vaccine evaluation application thereof |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311245855.6A Pending CN117265014A (en) | 2021-05-25 | 2021-05-25 | Construction method and application of virus S protein mutant |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN117265014A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134175A (en) * | 2021-11-01 | 2022-03-04 | 中国医学科学院输血研究所 | Packaging and detection method of SARS-CoV-2 pseudovirus system for simultaneously expressing RFP and luciferase |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005027963A2 (en) * | 2003-09-15 | 2005-03-31 | The United States Of America As Represented By Thesecretary Of Health And Human Services, Nih | METHODS AND COMPOSITIONS FOR THE GENERATION OF A PROTECTIVE IMMUNE RESPONSE AGAINTS SARS-CoV |
| US20100150923A1 (en) * | 2005-06-20 | 2010-06-17 | Chinese Academy Of Medical Sciences, Institute Of Basic Medical Sciences | Fusion proteins of recombinant sars coronavirus structural proteins, their production and uses |
| CN111593073A (en) * | 2020-03-18 | 2020-08-28 | 苏州奥特铭医药科技有限公司 | Double-reporter gene framework vector, four-plasmid pseudovirus packaging system and new packaging corolla pneumonia pseudovirus |
| CN111662884A (en) * | 2020-06-18 | 2020-09-15 | 中吉当康(北京)基因技术有限公司 | Pseudovirus, packaging method thereof and drug evaluation system |
| CN111676218A (en) * | 2020-03-12 | 2020-09-18 | 安徽省疾病预防控制中心（省健康教育所） | Full-length amplification sequencing method for spike gene of SARS-CoV-2 virus and primer thereof |
| CN112375768A (en) * | 2020-11-16 | 2021-02-19 | 同济大学 | Pseudo-virus of COVID-19 coronavirus, preparation method and application thereof |
| CN112618707A (en) * | 2020-10-15 | 2021-04-09 | 广州达博生物制品有限公司 | SARS-CoV-2 coronavirus vaccine and its preparation method |
-
2021
- 2021-05-25 CN CN202311245855.6A patent/CN117265014A/en active Pending
- 2021-05-25 CN CN202110573556.XA patent/CN113444745A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005027963A2 (en) * | 2003-09-15 | 2005-03-31 | The United States Of America As Represented By Thesecretary Of Health And Human Services, Nih | METHODS AND COMPOSITIONS FOR THE GENERATION OF A PROTECTIVE IMMUNE RESPONSE AGAINTS SARS-CoV |
| US20100150923A1 (en) * | 2005-06-20 | 2010-06-17 | Chinese Academy Of Medical Sciences, Institute Of Basic Medical Sciences | Fusion proteins of recombinant sars coronavirus structural proteins, their production and uses |
| CN111676218A (en) * | 2020-03-12 | 2020-09-18 | 安徽省疾病预防控制中心（省健康教育所） | Full-length amplification sequencing method for spike gene of SARS-CoV-2 virus and primer thereof |
| CN111593073A (en) * | 2020-03-18 | 2020-08-28 | 苏州奥特铭医药科技有限公司 | Double-reporter gene framework vector, four-plasmid pseudovirus packaging system and new packaging corolla pneumonia pseudovirus |
| CN111662884A (en) * | 2020-06-18 | 2020-09-15 | 中吉当康(北京)基因技术有限公司 | Pseudovirus, packaging method thereof and drug evaluation system |
| CN112618707A (en) * | 2020-10-15 | 2021-04-09 | 广州达博生物制品有限公司 | SARS-CoV-2 coronavirus vaccine and its preparation method |
| CN112375768A (en) * | 2020-11-16 | 2021-02-19 | 同济大学 | Pseudo-virus of COVID-19 coronavirus, preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 李琳 等: "中东呼吸综合征冠状病毒假病毒系统的建立及其在中和抗体检测中的应用", 《生物技术通讯》 * |
| 殷建国 等: "一种新型冠状病毒假病毒的制备及初步应用", 《免疫学杂志》 * |
| 程龙 等: "运用新型的反向PCR策略高效构建基因的点突变体", 《生物化学与生物物理进展》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134175A (en) * | 2021-11-01 | 2022-03-04 | 中国医学科学院输血研究所 | Packaging and detection method of SARS-CoV-2 pseudovirus system for simultaneously expressing RFP and luciferase |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117265014A (en) | 2023-12-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Corbet et al. | env sequences of simian immunodeficiency viruses from chimpanzees in Cameroon are strongly related to those of human immunodeficiency virus group N from the same geographic area | |
| Sato et al. | Development of a luminescence syncytium induction assay (LuSIA) for easily detecting and quantitatively measuring bovine leukemia virus infection | |
| Wright et al. | A robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and lyssaviruses in Africa | |
| Nelson et al. | Intrahost dynamics of human cytomegalovirus variants acquired by seronegative glycoprotein B vaccinees | |
| Brussel et al. | Use of a new RNA next generation sequencing approach for the specific detection of virus infection in cells | |
| Ntale et al. | Temporal association of HLA-B* 81: 01-and HLA-B* 39: 10-mediated HIV-1 p24 sequence evolution with disease progression | |
| Khan et al. | Report of the international conference on next generation sequencing for adventitious virus detection in biologicals | |
| Du et al. | Effects of mutations on replicative fitness and major histocompatibility complex class I binding affinity are among the determinants underlying cytotoxic-T-lymphocyte escape of HIV-1 gag epitopes | |
| Zhu et al. | Comparison of four methods using throat swabs to confirm rubella virus infection | |
| Willer et al. | Experimental infection of Cynomolgus Macaques (Macaca fascicularis) with human varicella-zoster virus | |
| Sodora et al. | Decreased levels of recent thymic emigrants in peripheral blood of simian immunodeficiency virus-infected macaques correlate with alterations within the thymus | |
| CN113444745A (en) | Construction of virus S protein mutant pseudovirus disc and vaccine evaluation application thereof | |
| Xia et al. | Neutralization of Omicron SARS-CoV-2 by 2 or 3 doses of BNT162b2 vaccine | |
| Su et al. | Reduced pathogenicity and transmission potential of Omicron BA. 1 and BA. 2 sublineages compared with the early severe acute respiratory syndrome coronavirus 2 D614G variant in Syrian hamsters | |
| Chin et al. | Molecular determinants of HIV-1 intersubtype recombination potential | |
| Singh et al. | Mutational characterization of Omicron SARS-CoV-2 lineages circulating in Chhattisgarh, a central state of India | |
| Depledge et al. | Native RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen | |
| CN118591621A (en) | Modified Vero cells and methods for their use in virus production | |
| CN113234832B (en) | Human EGFR gene missense mutation molecular marker and application thereof in prediction of drug resistance of targeted inhibitor | |
| US20220065869A1 (en) | Methods, Compositions, and Systems for Detecting Coronavirus Neutralizing Antibodies | |
| US11105805B2 (en) | Method for detecting HIV-1 co-receptor tropism | |
| Petrone et al. | Insights into the limited global spread of the immune evasive SARS-CoV-2 variant Mu | |
| Abavisani et al. | Investigating the mutations in the SARS-CoV-2 proteins among European countries | |
| US20100009341A1 (en) | Methods and means for assessing hiv gag/protease inhibitor therapy | |
| Basile et al. | Genomic evolution of SARS-CoV-2 variants of concern under in vitro neutralising selection pressure following two doses of the Pfizer-BioNTech BNT162b2 COVID-19 vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |